Apatite in-situ restoration

ABSTRACT

The present invention discloses methods of regenerating apatite surfaces, for example after purification of a target analyte.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 14/747,162, filed Jun. 23, 2015, which claimspriority to U.S. Provisional Application No. 62/015,894, filed Jun. 23,2014; and U.S. Provisional Application No. 62/082,017, filed on Nov. 19,2014, each of which is incorporated in its entirety herein for allpurposes.

BACKGROUND OF THE INVENTION

Apatite solid support surfaces, including hydroxyapatite, ceramicapatite, fluorapatite, and fluoride enhanced apatite, among otherapatite solid surfaces, are used for purification of a wide variety oftarget analytes. Apatite is most commonly utilized for purification ofbiological analytes, including proteins, carbohydrates, polynucleotides,and viral particles. Apatite possesses unique properties as apurification support because it provides affinity, cation, and anionexchange modalities in a single support. Apatite purification cangenerally be performed in two ways: (i) flow through purification; and(ii) bind and elute purification.

For flow through purification, traditionally, one (a) equilibrates thecolumn in a suitable buffer; (b) adds a sample to a column underconditions in which impurities bind to the column and the targetmolecule flows through and is collected, (c) cleans, or strips, thecolumn to remove adsorbed biological compounds with a cleaning/strippingsolution (e.g., a high molarity phosphate solution), and (d)regenerates, or sanitizes, the column with a strong alkaline hydroxidesolution so that the column can be re-used. In some cases, the strongalkaline hydroxide solution is replaced with a low molarity rinse forlong term storage or re-equilibration.

For bind and elute purification, traditionally, one (a) equilibrates thecolumn in a suitable buffer; (b) adds a sample to a column underconditions in which the target molecule binds to the column, (c) elutesthe target molecule (e.g., with a high molarity phosphate and/oralkaline halide solution), (d) cleans, or strips, the column to removeadsorbed biological compounds with a cleaning solution (e.g., a highmolarity phosphate solution), and (e) regenerates, or sanitizes, thecolumn with a strong alkaline hydroxide solution so that the column canbe re-used. In some cases, the strong alkaline hydroxide solution isreplaced with a low molarity rinse for long term storage orre-equilibration.

These traditional apatite purification methods can suffer from poorreproducibility and/or premature apatite deterioration. In some cases,this deterioration is due to the accumulation of hydronium ions (H3O+)on the apatite surface during exposure to equilibration, loading, orchromatography buffers. Hydronium ion accumulation can occur duringexposure to alkali metal salts at a pH of 8.0 or below. Hydronium ionaccumulation can also occur during exposure to phosphate buffers at a pHof less than about 6.5. Other buffer compositions can also causehydronium ion accumulation. These hydronium ions are then desorbed uponexposure to a subsequent buffer, such as an elution buffer (e.g., duringbind and elute purification) or a cleaning/stripping buffer (e.g., afterflow through purification). This desorption causes the resin todeteriorate over time, resulting in a loss of resin mass and/or adecline in the particle strength of the resin.

BRIEF SUMMARY OF THE INVENTION

The present inventor has discovered that the deterioration of an apatitesolid surface during, or subsequent to, a chromatographic procedure forpurifying a target molecule from a sample can be surprisingly reduced,eliminated, or reversed by treating the apatite solid surface with abuffered calcium solution, followed by a phosphate buffered solution,followed by an alkaline hydroxide. The buffered calcium solution,phosphate buffered solution, and alkaline hydroxide can be appliedsubsequent to a bind and elute or flow through purification procedure.

In one aspect, the present invention provides a method of purifying atarget analyte with an apatite solid surface, the method comprising: (a)contacting the apatite solid surface with the target analyte, therebyseparating the target analyte from one or more contaminants; (b)collecting the target analyte; and (c) regenerating the apatite solidsurface the regenerating comprising, (i) contacting the apatite solidsurface with a buffered calcium solution comprising a calcium ion at aconcentration of at least about 5 mM and a buffer, wherein the ratio ofbuffer concentration to calcium ion concentration is at least about 1,1.5, or 2, and the pH of the solution is at least about 5, 5.1, 5.2,5.3, 5.4, or 5.5; (ii) contacting the apatite solid surface with aphosphate buffered solution at a pH of at least about 6.5; and (iii)contacting the apatite solid surface with a solution comprising anhydroxide. In one embodiment, the buffer is a zwitterionic buffer. Inone embodiment, the buffer is a phosphate buffer.

In one embodiment, the present invention provides a method of purifyinga target analyte with an apatite solid surface, the method comprising:(a) contacting the apatite solid surface with the target analyte,thereby separating the target analyte from one or more contaminants; (b)collecting the target analyte; and (c) regenerating the apatite solidsurface the regenerating comprising, (i) contacting the apatite solidsurface with a buffered calcium solution comprising a calcium ion at aconcentration of at least about 10 mM and a zwitterionic buffer, whereinthe ratio of zwitterionic buffer concentration to calcium ionconcentration is at least about 2, and the pH of the solution is atleast about 6.5; (ii) contacting the apatite solid surface with aphosphate buffered solution at a pH of at least about 6.5; and (iii)contacting the apatite solid surface with a solution comprising anhydroxide.

In one embodiment, the present invention provides a method wherein (a)comprises binding the target analyte to the apatite solid surface, and(b) comprises eluting the target analyte from the apatite solid surface.In another embodiment, (a) comprises contacting the apatite solidsurface to the target analyte, thereby flowing the target analytethrough the apatite solid surface, and (b) comprises collecting thetarget analyte in the flow through.

In some cases, the zwitterionic buffer is a sulfonic acid containingbuffer. In some cases, the sulfonic acid containing buffer is MES,PIPES, ACES, MOPSO, MOPS, BES, TES, HEPES, DIPSO, TAPS, TAPSO, POPSO, orHEPPSO, EPPS, CAPS, CAPSO, or CHES. In some cases, the sulfonic acidcontaining buffer is MES.

In one embodiment, the calcium ion is at least about 1 mM, 2 mM, 3 mM, 4mM, 5 mM, 10 mM (e.g., 10.1 mM, 10.2 mM, 10.3 mM, 10.4 mM, or 10.5 mM),20 mM, 25 mM or at least about 50 mM. In another embodiment, the ratioof buffer concentration (e.g., zwitterionic buffer concentration) tocalcium ion concentration is at least about 2.5, 3, or 4. In yet anotherembodiment, the buffered calcium solution comprises calcium chloride orcalcium nitrate. In yet another embodiment, the phosphate bufferedsolution comprises a solution containing from about 0.1 M or 0.2 M toabout 1.0 M phosphate, or from about 0.1 M or 0.2 M to about 0.5 Mphosphate, at a pH of from about 6.5 to about 8. In some cases, thephosphate buffered solution comprises 400 mM phosphate at a pH of 7.0.

In one embodiment, the hydroxide comprises an alkaline hydroxide. Insome cases, the alkaline hydroxide comprises sodium or potassiumhydroxide. In one embodiment, the regenerating reverses or eliminatesdegradation of the column that occurs during protein purification orcolumn cleaning steps. In another embodiment, the regenerating increasesthe strength of the apatite solid surface by at least about 1%, 5%, 10%,15%, 20%, or more.

In one embodiment the regenerating is performed before, or replaces, aphosphate cleaning/stripping step that elutes adsorbed biologicalcompounds. In some cases, the regenerating step is performed afterelution of target analyte.

In one embodiment, the (ii) contacting the apatite solid surface with asolution comprising phosphate at a pH of at least about 6.5 furthercomprises: contacting the apatite solid surface with a solutioncomprising phosphate at a concentration of 10 mM, or less than about 10mM, at a pH of at least about 6.5 or 7; and then contacting the apatitesolid surface with a solution comprising phosphate at a concentration ofat least about 100 mM, 200 mM, 400 mM, or 500 mM, at a pH of at leastabout 6.5 or 7.

In one embodiment, the regenerating consists of (i), a wash, (ii), and(iii).

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1: depicts results from performing the apatite purification profileof Example 7, Table X.

FIG. 2: depicts results from performing the purification profile ofExample 7, Table X, after application of indicated in situ regenerationsolutions.

FIG. 3: depicts results from performing the purification profile ofExample 7, Table X, after application of indicated in situ regenerationsolutions.

FIG. 4: depicts results from performing the purification profile ofExample 7, Table X, after application of indicated in situ regenerationsolutions.

FIG. 5: depicts results from performing the purification profile ofExample 7, Table X, after application of indicated in situ regenerationsolutions.

DEFINITIONS

“Apatite” refers to a mineral of phosphate and calcium of the generalformula Ca₅(PO₄)₃(X), wherein X is a negatively charged ion. Generally,X is F, Cl, or OH. However, the structure and chemistry of apatite allowfor numerous substitutions, including a variety of metal cations (e.g.,one or more of K, Na, Mn, Ni, Cu, Co, Zn, Sr, Ba, Pb, Cd, Sb, Y, U, orvarious rare earth elements) that substitute for Ca in the structure,and anionic complexes (e.g., AsO₄ ⁻³, SO₄ ⁻², CO₃ ⁻², SiO₄ ⁻⁴, etc.)that substitute for PO₄ ⁻³.

“Hydroxyapatite” refers to a mixed mode solid support comprising aninsoluble hydroxylated mineral of calcium phosphate with the structuralformula Ca₁₀(PO₄)₆(OH)₂. Its dominant modes of interaction arephosphoryl cation exchange and calcium metal affinity. Hydroxapatite iscommercially available in various forms, including but not limited toceramic, crystalline and composite forms. Composite forms containhydroxyapatite microcrystals entrapped within the pores of agarose orother beads.

“Fluorapatite” refers to a mixed mode support comprising an insolublefluoridated mineral of calcium phosphate with the structural formulaCa₁₀(PO₄)₆F₂. Its dominant modes of interaction are phosphoryl cationexchange and calcium metal affinity. Fluorapatite is commerciallyavailable in various forms, including but not limited to ceramic andcrystalline composite forms.

An “apatite solid surface” refers to fused nanocrystals (ceramicapatite), microcrystals, or compounded microcrystals of apatite. Apatitesolid surfaces include, but are not limited to, hydroxyapatite, orfluorapatite. Ceramic apatites include, but are not limited to, ceramichydroxyapatite (e.g., CHT™) or ceramic fluorapatite. Ceramic apatitesare a form of apatite minerals in which nanocrystals are agglomeratedinto particles and fused at high temperature to create stable ceramicmicrospheres suitable for chromatography applications. Compoundedmicrocrystals include but are not limited to HA Ultragel® (Pall Corp.).Microcrystals include but are not limited to Bio-Gel HTP, Bio-Gel® HT,DNA-Grade HT (Bio-Rad) and Hypatite C (Clarkson Chromatography).

“Sample” refers to any composition having a target molecule or particleof interest. A sample can be unpurified or partially purified. Samplescan include samples of biological origin, including but not limited toblood, or blood parts (including but not limited to serum), urine,saliva, feces, as well as tissues. Samples can be derived fromunpurified, partially purified, or purified cell lysate or spent cellgrowth media.

Deterioration of a resin that occurs upon use can cause the resinparticles to lose their strength and thus to break apart into smallerparticles causing blockage in the column. The deterioration can occur asa chemical breakdown of the apatite, causing a loss of mass which can inturn result in a loss of column volume, a loss in particle strength, anincrease in particle breakage, or a combination thereof. In someembodiments of the present invention, such effects can be reversed bythe present invention. The reversal of deterioration that can beachieved by the practice of the present invention can result in a lowerrate of resin mass loss, a lower rate of decline in particle strength,or both. In many cases, the reversal of deterioration can be accompaniedby increases in resin mass, particle strength, or both.

Mass of the apatite solid surface can be assayed by, e.g., weighing adried apatite sample, for example after washing away buffer componentsand adsorbed biological compounds. Apatite media strength can be assayedby, e.g., measuring resistance to agitational force (e.g., stirring),resistance to sonication, or resistance to compression (e.g.,application of a uniaxial compressive force). Resistance to sonicationor agitational force can be measured by inspection of the apatite solidsurface after the treatment to measure the generation of fines.Resistance to compression can be measured by measuring the forcerequired to compress a given mass of apatite to a constant terminalforce setting and determining the compressed distance. Apatitedeterioration or degradation can be measured relative to a sample thathas not been subjected to an apatite purification (i.e., purification ofa target molecule using apatite) or an apatite regeneration procedure.

An “alkaline hydroxide” refers to a metal alkali hydroxide comprisingany cation elements in Group I of the periodic table, including, e.g.,lithium (Li), sodium (Na), potassium (K), rubidium (Rb), cesium (Cs),and francium (Fr). Thus, exemplary alkaline hydroxides include, forexample, NaOH, LiOH, and KOH.

A zwitterionic buffer is a buffer that can contain a formal positive anda formal negative electrical charge at the same time. Exemplaryzwitterionic buffers can include, but are not limited to, bufferscontaining a sulfonic acid group. As used herein, a “sulfonic acid”refers to a member of the class of organosulfur compounds with thegeneral formula RS(═O)₂—OH, where R is an organic group (e.g., alkyl, oralkene, or aryl) and the S(═O)₂—OH group is a sulfonyl hydroxide.

Exemplary zwitterionic buffers containing a sulfonic acid group caninclude, but are not limited to, aminoalkanesulfonic acids. Exemplaryaminoalkanesulfonic acids can include, but are not limited to,aminoalkanesulfonic acids with a minimum of two carbons between amineand sulfonic acid groups. Exemplary zwitterionic buffers containing asulfonic acid group can include, but are not limited to,N,N-dialkylaminomethanesulfonic acids.

Exemplary zwitterionic buffers containing a sulfonic acid group caninclude, but are not limited to, MES (2-(N-morpholino)ethanesulfonicacid), PIPES (1,4-Piperazinediethanesulfonic acid), ACES(2-(carbamoylmethylamino)ethanesulfonic acid), MOPSO(3-morpholino-2-hydroxypropanesulfonic acid), MOPS(3-morpholinopropane-1-sulfonic acid), BES(N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid), TES(2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonicacid), HEPES (2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid),DIPSO (3-(N,N-Bis[2-hydroxyethyl]amino)-2-hydroxypropanesulfonic acid),TAPS(3-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]propane-1-sulfonicacid), TAPSO(3-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]-2-hydroxypropane-1-sulfonicacid), POPSO (piperazine-1,4-bis(2-hydroxypropanesulfonic acid)), orHEPPSO (N-(2-Hydroxyethyl)piperazine-N′-(2-hydroxypropanesulfonicacid)), EPPS (N-(2-Hydroxyethyl)piperazine-N′-(3-propanesulfonic acid)),CAPS (3-(Cyclohexylamino)-1-propanesulfonic acid), CAPSO(N-cyclohexyl-2-hydroxyl-3-aminopropanesulfonic acid), CHES(2-(Cyclohexylamino)ethanesulfonic acid), MOBS(4-(N-morpholino)butanesulfonic acid), TABS(N-tris(hydroxymethyl)-4-aminobutanesulfonic acid), or AMPSO(N-(1,1-Dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid).

Calcium ion for use as a restoration material in the proceduresdescribed herein can be supplied by calcium hydroxide or by a solublecalcium salt, typically a salt that is soluble in water. Calcium halidesand calcium nitrate are examples of calcium salts that can be used. Anexemplary calcium halide is calcium chloride. In some cases, calciumchloride is preferred when the chromatography buffer during flow throughpurification, or the elution buffer during bind and elute purification,contains an alkali metal chloride.

As used herein, the terms “buffer,” “buffered,” and the like, in thecontext of a buffered calcium solution refers to a buffer that iscompatible with (e.g., does not substantially interact with orprecipitate in complex with) calcium under the specified conditions andis employed for the purpose of stabilizing the pH of an aqueous solutionat or near a specified value, or within a specified range. As such,generally, the “buffer” in a buffered calcium solution cannot be water.In some embodiments, the “buffer” in a buffered calcium solution is notphosphate. In some embodiments, the “buffer” in a buffered calciumsolution is phosphate. In some embodiments, the “buffer” in a bufferedcalcium solution used in an in situ regeneration protocol (e.g., after(e.g., immediately after) elution or target analyte flow through, orafter (e.g., immediately after) elution or target analyte flow throughand a wash) does not contain an alkali metal salt (e.g., sodiumchloride), or contains less than about 0.1, 0.05, or 0.01 M alkali metalsalt.

Phosphate can be used in a variety of buffers for apatite equilibration,chromatography, elution, cleaning/stripping, or apatite regeneration.Phosphate can be supplied from any soluble phosphate salt, typically asalt that is soluble in water. Alkali metal or alkaline earth metalphosphates are examples, with sodium or potassium phosphate asparticularly convenient examples. Alkali or alkaline earth metalphosphate salts can be utilized in mono- and di-basic forms, or acombination thereof.

As used herein, the term “about” refers to the recited number and anyvalue within 10% of the recited number. Thus, “about 5” refers to anyvalue between 4.5 and 5.5, including 4.5 and 5.5.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

Traditional apatite protein purification procedures generally either donot protect the apatite solid surface from deterioration, or seek toprevent deterioration. Methods that seek to prevent deteriorationinclude the use of one or more common ions (e.g., U.S. application Ser.No. 13/205,354), or the use of a high pH phosphate solution (e.g., U.S.application Ser. No. 10/327,495). The presence of an ionic species inthe buffer that is common to a component of the apatite solid surface (acommon ion) can suppress leaching of that component from the apatitesolid surface. Thus, calcium and/or phosphate buffers are oftenpreferred during apatite equilibration, loading, flow through, elution,or cleaning/stripping. A high pH phosphate solution can function as acommon ion, as well as minimizing potentially damaging pH excursions.

Other methods that seek to prevent deterioration include neutralizationof accumulated hydronium ions prior to their release from the apatitesolid surface during the apatite purification procedure. Neutralizationcan be performed with a strong base, such as an alkaline hydroxide(e.g., U.S. application Ser. No. 13/363,670). Neutralization can also beperformed with a basic amino compound or sulphonated amine compound(e.g., U.S. application Ser. No. 13/006,022). Accumulation of hydroniumions on the apatite surface can occur due to a variety of mechanismsduring equilibration, loading, flow through, and washing steps.

In particular, the presence of alkali metal salts can increase, orpromote, accumulation of hydronium ions. A high pH phosphate solution(e.g., phosphate at a pH of about 6.5 or higher) of sufficientconcentration (e.g., 100, 200, 300, 400 mM, or higher), can providebuffering capacity to mitigate the pH shift that commonly occurs duringhydronium ion release, and therefore reduce acid solubilization of theapatite. The use of alkali metal salts concurrently with a phosphatebuffer of at a suitable pH and concentration generally mitigates massloss to a significant degree. However, media strength can still besignificantly decreased. Neutralization of accumulated hydronium ionscan reduce the amount of accumulated hydronium ions, and thus reducedegradation during a subsequent phosphate buffer cleaning step.

The present invention is based on the surprising discovery that anapatite solid surface can be significantly regenerated by treating witha buffered calcium regeneration solution. Generally, the bufferedcalcium solution is applied after the target molecule has been purifiedand collected. In some cases, the buffered calcium solution is appliedafter the apatite solid surface has been cleaned/stripped (e.g., with ahigh molarity phosphate buffer, such as 100, 200, 300, 400, or 500 mMphosphate, or higher) and/or sanitized (e.g., with alkaline hydroxide ata concentration of about 0.1, 0.5, or 1 M). The buffered calciumsolution can be optionally washed away. In some cases, the apatite isthen treated with a phosphate buffered solution (e.g., a phosphatebuffered solution that does not contain calcium). In some cases, thephosphate buffered solution applied after the buffered calcium solutioncontains a higher concentration of phosphate than in the bufferedcalcium solution. In some cases, the order of the buffered calciumregeneration solution and the phosphate buffered regeneration solutionis reversed.

After contacting the phosphate buffered solution with the apatite solidsurface, the apatite solid surface can be further treated with anhydroxide. The regeneration procedures described herein (e.g.,contacting apatite with a buffered calcium solution, then phosphatebuffered solution, and then alkaline hydroxide; or contacting apatitewith a phosphate buffered solution, then a buffered calcium solution,and then alkaline hydroxide) provide a substantial and surprising degreeof regeneration. This substantial and surprising degree of regenerationcan be indicated as a reduction, elimination, or reversal ofdegradation, as measured by change (e.g., loss) in apatite mass or lossin apatite strength. In some cases, regeneration can be indicated as amaintenance, or decrease in loss of chromatographic resolution orselectivity. In some cases, the regeneration methods described hereincan be combined with one or more methods that reduce or preventdegradation, such as those described in the paragraphs above.

Selectivity can be measured as a ratio of retention volumes (e.g.,adjusted for dead volume) between two different components in a mixture.Resolution can be measured as the ratio of the distance between twochromatographic peak maxima to the mean value of the peak width at baseline between two different components in a mixture. In some cases, thecomponents are protein chromatography standards. Exemplary proteinchromatography standards include, but are not limited to, ovalbumin,myoglobin, alpha chymotrypsinogen a, or cytochrome c.

II. Methods

Described herein, are apatite regeneration methods for reducing,eliminating, or reversing apatite deterioration by treating the apatitesolid surface with a buffered calcium solution, followed by a phosphatebuffered solution, followed by an alkaline hydroxide. The bufferedcalcium solution, phosphate buffered solution, and alkaline hydroxidecan be applied subsequent to a bind and elute or flow throughpurification procedure.

In some embodiments, a sample is contacted with an apatite solid surface(e.g., an equilibrated apatite solid surface), the target molecule iscollected (e.g., during flow through purification, or after elution),and the apatite is regenerated by contacting the apatite solid surfacewith a buffered calcium solution, followed by a phosphate bufferedsolution, followed by an alkaline hydroxide. In some cases, the apatitesolid surface is used multiple times for target analyte purificationprior to application of one or more regeneration steps described herein.

In some embodiments, the apatite solid surface is washed or rinsed priorto regenerating. In other embodiments, the apatite solid surface is notwashed or rinsed prior to regenerating. In some cases, the resin istreated with a wash solution to remove any excess calcium, phosphate, orhydroxide ions. One of skill in the art can readily select a suitablewash buffer. Generally, the wash buffer can be at a pH, composition, andconcentration that does not substantially leach components of theapatite surface, release accumulated hydronium ions, or generateundesirable precipitate. For example, the wash buffer can be compatible,and thus not precipitate when mixed, with the preceding and subsequentbuffer. Suitable washing buffers can include buffer compositionstypically used for equilibration, loading, or flow through of apatite.In some cases, the apatite solid surface is washed with a low molarityphosphate buffer (e.g., phosphate at a concentration of less than about100 mM, 50 mM, 25 mM, 20 mM, 15 mM, 10 mM, or 5 mM). The pH of the washbuffer can be at least about 5, 5.1, 5.2, 5.3, or 5.4, at least about5.5, at least about 6, or at least about 6.5, 7, or 8. An exemplary washbuffer pH is 5.5, 6, or 6.5. In some cases, a water wash is applied, andthe amounts can vary widely. A typical water wash will be at least about0.2 resin volumes, and in most cases from about 0.2 to about 1.5 or fromabout 0.2 to about 2 resin volumes.

The apatite solid surface can then be regenerated. In some cases, theapatite solid surface can be regenerated, e.g., after elution, afterflow through, after neutralization, after cleaning/stripping, afterrinsing, or after storage. In some cases, the apatite solid surface canbe regenerated after a wash, e.g., after application of a wash buffer toremove a flow through, elution, neutralization, rinsing, storage, orcleaning/stripping buffer.

A. Buffered Calcium Solution

The regeneration begins with contacting the apatite solid surface with abuffered calcium solution. Although, regeneration of the apatite solidsurface has been attempted using an unbuffered calcium solution, thepresent inventors have found that the use of a buffered calcium solutionappears to significantly and surprisingly enhance the degree ofregeneration obtained. The calcium ion concentration of the bufferedcalcium solution and the amount of the buffered calcium solution passedthrough the resin can vary, but will generally be selected as any amountthat will reduce, eliminate, or reverse the deterioration of the resinthat occurs during apatite use (e.g., during purification, duringelution, or during cleaning/stripping).

Without wishing to be bound by theory, it is believed that the bufferedcalcium solution interacts with the apatite solid surface to generate aloosely bound (e.g., non-covalent) calcium layer on the apatite solidsurface. In some cases, this calcium layer replaces some or all (or morethan all) of the calcium lost during previous purification steps. Thus,an amount, volume, concentration, etc. of calcium ion, or any othercomponent or aspect of the buffered calcium solution that will reduce,eliminate, or reverse the deterioration of the resin that occurs duringapatite use, can be an amount that allows for sufficient formation of aloosely bound calcium layer.

The calcium ion concentration is generally selected to be below thesolubility limit of calcium at the pH and temperature of the bufferedcalcium solution. Moreover, the concentration can vary based on thepresence, absence, or concentration of other components in the bufferedcalcium solution, such as the selected buffering agent, or based on theselected composition of any preceding buffer. In certain embodiments ofthe concepts herein, best results will be achieved with a calcium ionconcentration of from about 5 mM, 5.1 mM, 5.2 mM, 5.3 mM, 5.4 mM, 5.5mM, 5.6 mM, 5.7 mM, 5.8 mM, 5.9 mM, 6 mM, 6.5 mM, 7, mM, 8 mM, 9 mM, 10mM, 10.1 mM, 10.2 mM, 10.3 mM, 10.5 mM, or 11 mM to about 15 mM, 20 mM,25 mM, 30 mM, 40 mM, 50 mM, 75 mM, 100 mM, or 250 mM. In certainembodiments, the calcium ion concentration in the buffered calciumsolution is from about 5 mM to about 10 mM, from about 5 mM to about 25mM, from about 20 mM to about 100 mM, or from about 25 mM to about 50-75mM, including 5 mM, 5.1 mM, 5.2 mM, 5.3 mM, 5.4 mM, 5.5 mM, 5.6 mM, 5.7mM, 5.8 mM, 5.9 mM, 6 mM, 6.5 mM, 7, mM, 8 mM, 9 mM, 10 mM, 10.1 mM,10.2 mM, 10.3 mM, 10.5 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 20 mM, 25mM, 30 mM, 40 mM, 60 ppm, 70 mM, 80 mM, 90 mM, 110 mM, 150 mM, 200 mM,300 mM, or higher.

The volume of the solution needed to achieve the restoration can varywith the calcium ion concentration, but in most cases best results willbe achieved with from about 1.0 to about 10.0 resin volumes of solution,and in many cases with about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8,1.9, or about 2 resin volumes. In some cases, the volume can be up toabout 6 resin volumes, including 2, 3, 4, or 5 resin volumes. In somecases, the volume is less than 3 column volumes. In some cases, a highcalcium ion concentration at a volume that is less than a resin volume(e.g., less than about 0.9, 0.7, 0.5 volumes) can be utilized.

A wide variety of buffers are suitable for the buffered calcium solutionfor apatite regeneration. In some embodiments, a buffer for the bufferedcalcium solution that does not appreciably form metal complexes insolution (e.g., does not form a complex with calcium at the pH of thebuffer solution) can comprise the buffer component of the bufferedcalcium solution. In some embodiments, a buffer that does not containprimary or secondary (i.e., R₂—N, wherein R is not H) amine can comprisethe buffer component of the buffered calcium solution. In someembodiments, a zwitterionic buffer is preferred. In some embodiments, abuffer (e.g., a zwitterionic buffer) that contains a sulfonic acidmoiety is preferred. In some embodiments, a buffer (e.g., a zwitterionicbuffer) that contains a sulfonic acid and a tertiary amine (i.e., R₃—N,wherein R is not H) is preferred. Exemplary zwitterionic bufferssuitable for use as a buffering agent in the buffered calcium solutioninclude, but are not limited to, one or more of the following: MES,PIPES, ACES, MOPSO, MOPS, BES, TES, HEPES, DIPSO, TAPS, TAPSO, POPSO, orHEPPSO, EPPS, CAPS, CAPSO, CHES, MOBS, TABS, or AMPSO. In someembodiments, the buffer of the buffered calcium solution contains aprimary, secondary, or a tertiary amine. In some embodiments, the bufferof the buffered calcium solution contains a primary, secondary or atertiary amine and a one or more carboxylate or hydroxymethyl groups. Insome embodiments, the buffer of the buffered calcium solution istricine, bicine, or tromethamine. In some embodiments, the buffer of thebuffered calcium solution isbis(2-hydroxyethyl)-amino-tris(hydroxymethyl)-methane), or1,3-bis(tris(hydroxymethyl) methylamino) propane.

The buffer concentration in the buffered calcium solution can vary, butwill generally be selected as a concentration that is at least as highas the calcium ion concentration of the solution. Moreover, theconcentration can vary based on the selected buffering agent, or theselected composition of any preceding buffer. Thus, the ratio of thebuffer concentration to the calcium ion concentration is generally atleast about 1, e.g., 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2,2.1, 2.2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, or higher. Generally, the bufferconcentration is also selected such that it is below the solubilitylimit of the buffering agent. In some cases, preferred buffering agentsinclude those that have a high solubility limit.

The pH of the buffered calcium solution can vary, but will generally beselected as any amount that will reduce, eliminate, or reversedeterioration of the resin that occurs during apatite use (e.g., duringpurification, during elution, or during cleaning/stripping). Moreover,the pH can vary based on the selected apatite solid surface, theselected buffering agent, the selected concentration of one or morecomponents, or the selected composition of any preceding buffer.Typically, the pH is, or is at least about, 5, 5.1, 5.2, 5.3, 5.4, 5.5,5.6, 6, 6.2, 6.5, 7, 7.5, or 8. In some embodiments, the pH is, or is atleast about, 5.5, 6, 6.5, 7, 7.5, or 8. In some embodiments, the pH is5.5, 6, 6.5, 7, 7.5, or 8. In some embodiments, the pH is 5.1, 5.2, 5.3,or 5.4. In some cases, the pH is 5.3. In some cases, the pH is 5.5, 5.6,5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1,7.2, 7.3, 7.4, or 7.5. In some cases, the pH is 7.0. In some cases, thepH is 5.6. In some cases, the pH is 6.2. In some cases, the pH is 5.4.

In some embodiments, the buffer of the buffered calcium solution is aphosphate buffer. In such cases, the calcium and phosphateconcentrations and the pH of the solution can be selected to provideregeneration while avoiding precipitant formation, or avoiding asupersaturated solution. For example, the pH of the phosphate bufferedcalcium solution can be selected to be sufficiently low (e.g., a pH ofabout, or less than about, 6.5, 6.4, 6.3, 6.2, 6.1, 6, 5.9, 5.8, 5.7,5.6, 5.5, 5.4, 5.3, 5.2, 5.1, or 5). In some cases, the pH is 5.1, 5.2,5.3, 5.4, or 5.5. In some cases, the pH is 5.3. As another example, thecalcium concentration of the phosphate buffered calcium solution can beabout, or less than about, 50 mM, 40 mM, 35 mM, 30 mM, 25 mM, 20 mM, 15mM, 10 mM, 7 mM, 6 mM, 5.9 mM, 5.8 mM, 5.7 mM, 5.6 mM, 5.5 mM, 5.4 mM,5.3 mM, 5.2 mM, 5.1 mM, or 5 mM. In some cases, the calciumconcentration of the phosphate buffered solution is, or is about, 15 mM,14 mM, 13 mM, 12 mM, 11 mM, 10.5 mM, 10.4 mM, 10.3 mM, 10.2 mM, 10.1 mM,10 mM, or 9.5 mM. In some cases, the calcium concentration is 10 or 10.2mM. In some cases, the calcium concentration is 10 mM. As anotherexample, the phosphate concentration of the phosphate buffered calciumsolution can be about, or less than about, 50 mM, 40 mM, 35 mM, 30 mM,29 mM, 28 mM, 27 mM, 26 mM, 25 mM, 24 mM, 23 mM, 21 mM, 20 mM, 18 mM, 17mM, 16 mM, or 15 mM. In some cases, the use of a phosphate bufferedcalcium solution provides regeneration with or without a preceeding orsubsequent high molarity (e.g., 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,0.9, or 1 M) phosphate buffer step.

In some embodiments, the apatite solid surface is in a column, e.g., achromatography column, and the buffered calcium solution can be appliedto the apatite solid surface at a flow rate. The flow rate can vary, butwill generally be selected as any rate that will reduce, eliminate, orreverse deterioration of the resin that occurs during apatite use (e.g.,during purification, during elution, or during cleaning/stripping).Suitable flow rates, include rates that are typically used duringequilibration, loading, elution, cleaning/stripping, sanitation, orrinsing of apatite. An exemplary flow rate is 400 cm/hr. In some cases,the flow rate is substantially lower than 400 cm/hr (e.g., 300, 200,100, or 50 cm/hr, or less). The use of a low flow rate can allow agreater contact time between the apatite solid surface and the bufferedcalcium solution. A low flow rate can be particularly preferred when theconcentration of calcium or buffering agent, or the volume of thebuffered calcium solution, is low. A low flow rate can also be preferredwhen the buffered calcium solution, or the preceding solution, isviscous or the column is fouled with adsorbed biological compounds.Alternatively, the flow rate can be higher than 400 cm/hr. In somecases, the formation of a loosely bound layer of calcium is rapid and ahigh flow rate can advantageously reduce the time required for apatiteregeneration.

In some embodiments, the apatite solid surface is contacted with thebuffered calcium solution in a batch format. In a batch format, thebuffered calcium solution can be applied by pouring the buffered calciumsolution onto the apatite solid surface, or pouring a slurry of theapatite solid surface into the buffered calcium solution. The contacttime can vary, but will generally be selected as any time that willreduce, eliminate, or reverse deterioration of the resin that occursduring apatite use (e.g., during purification, during elution, or duringcleaning/stripping).

In some embodiments, the apatite solid surface is then washed or rinsed.One of skill in the art can readily select a suitable wash buffer. Insome cases, the resin is treated with a wash solution between theindividual regeneration treatments to remove any excess calcium,phosphate, or hydroxide ions. Generally, the wash buffer can be at a pH,composition, and concentration that does not substantially leachcomponents of the apatite surface, release accumulated hydronium ions,or generate undesirable precipitate. For example, the wash buffer can becompatible, and thus not precipitate when mixed, with the preceding andsubsequent buffer. As another example, the wash buffer can be selectedthat does not leach any loosely bound calcium layer formed during thecontacting of the apatite solid surface with the buffered calciumsolution. Suitable washing buffers can include buffer compositionstypically used for equilibration, loading, or flow through of apatite.In some cases, the apatite solid surface is washed with a low molarityphosphate buffer (e.g., phosphate at a concentration of less than about100 mM, 50 mM, 25 mM, 20 mM, 15 mM, 10 mM, or 5 mM). The pH of the washbuffer can be at least about 5, at least about 5.5, at least about 6, orat least about 6.5, 7, or 8. In some cases, a water wash is applied, andthe amounts can vary widely. A typical water wash will be at least about0.2 resin volumes, and in most cases from about 0.2 to about 1.5 or fromabout 0.2 to about 2 resin volumes.

B. Phosphate Buffered Solution

The apatite solid surface can then be contacted with a phosphatecontaining buffer after the apatite has been contacted with a bufferedcalcium solution. Alternatively, the phosphate containing buffer can becontacted with apatite before the apatite has been contacted with abuffered calcium solution. In some cases, an intervening wash step isapplied between the buffered calcium solution and the phosphatecontaining buffer. The phosphate concentration of the phosphatecontaining buffer and the amount of the phosphate containing bufferpassed through the resin can vary, but will generally be selected as anyamount that will reduce, eliminate, or reverse the deterioration of theresin that occurs during apatite use (e.g., during purification, duringelution, or during cleaning/stripping). Without wishing to be bound bytheory, it is believed that the phosphate containing buffer interactswith the apatite solid surface, or a loosely bound calcium layer formedduring contact with the buffered calcium solution, to generate a looselybound (e.g., non-covalent) phosphate layer on the apatite solid surface.In some cases, this phosphate layer replaces some or all (or more thanall) of the phosphate lost during previous purification steps. Thus, anamount, volume, concentration, etc. of phosphate, or any other componentor aspect of the phosphate containing buffer that will reduce,eliminate, or reverse the deterioration of the resin that occurs duringapatite use, can be an amount that allows for sufficient formation of aloosely bound phosphate layer.

The phosphate concentration of the phosphate containing buffer isgenerally selected to be below the solubility limit of the phosphate atthe pH and temperature of the buffer. Moreover, the concentration canvary based on presence or absence of other components of the buffer, orthe selected composition of any preceding buffer. In certain embodimentsof the concepts herein, best results will be achieved with a phosphateconcentration of from about 10 mM to about 1, 1.5, or 2 M; from about 20mM to about 1.5 M; or from about 25 mM to about 1 M; from about 50 mM toabout 1 M; including at least about, or about, 10 mM, 15 mM, 20 mM, 25mM, 30 mM, 40 mM, 50 mM, 60 ppm, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM,150 mM, 200 mM, 300 mM, 500 mM, 750 mM, 1 M, or higher. In some cases,the phosphate concentration is 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 40 mM,50 mM, 60 ppm, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, 150 mM, 200 mM, 300mM, 500 mM, 750 mM, 1 M, or higher. In some cases, the phosphateconcentration is from, or from about, 0.1 or 2 M to, or to about, 0.4 M,0.5 M, or 1 M. In some cases, the column is contacted with a lowconcentration phosphate buffer (e.g., 2, 5, 10, 15, 20, or 25 mM) andthen a high concentration phosphate buffer (e.g., 30; 50; 75; 100; 250;500; 750; 1,000; 1,500; or 2,000 mM). In some cases, the use of a lowconcentration phosphate buffer followed by a high concentrationphosphate buffer can avoid potential incompatibility (e.g.,precipitation) between the buffered calcium solution and the highconcentration phosphate buffer.

The pH of the phosphate containing buffer and the amount of thephosphate containing buffer passed through the resin can vary, but willgenerally be selected as any pH that will reduce, eliminate, or reversethe deterioration of the resin that occurs during apatite use (e.g.,during purification, during elution, or during cleaning/stripping).Exemplary pH values suitable for apatite regeneration with a phosphatecontaining buffer include any pH that is at least about 5, at leastabout 5.5, at least about 6, at least about 6.5, at least about 7, atleast about 7.5, at least about 8, or at least about 8.5, or higher. Insome cases, the pH of the phosphate containing buffer is 5, 5.5, 6, 6.5,7, 7.5, 8, 8.5, 9, 9.5, 10, or higher.

The volume of the solution needed to achieve the restoration can varywith the phosphate ion concentration, but in most cases best resultswill be achieved with from about 1.0 to about 10.0 resin volumes ofsolution, and in many cases with about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6,1.7, 1.8, 1.9, or about 2 resin volumes. The volume can be up to about 6resin volumes, including 2, 3, 4, or 5 resin volumes. In some cases, ahigh phosphate concentration at a volume that is less than a resinvolume (e.g., less than about 0.9, 0.7, 0.5 volumes) can be utilized.

In some embodiments, the apatite solid surface is in a column, e.g., achromatography column, and the phosphate containing buffer can beapplied to the apatite solid surface at a flow rate. The flow rate canvary, but will generally be selected as any rate that will reduce,eliminate, or reverse deterioration of the resin that occurs duringapatite use (e.g., during purification, during elution, or duringcleaning/stripping). Suitable flow rates, include rates that aretypically used during equilibration, loading, elution,cleaning/stripping, sanitation, or rinsing of apatite. An exemplary flowrate is 400 cm/hr. In some cases, the flow rate is substantially lowerthan 400 cm/hr (e.g., 300, 200, 100, or 50 cm/hr, or less). The use of alow flow rate can allow a greater contact time between the apatite solidsurface and the phosphate containing buffer. A low flow rate can beparticularly preferred when the concentration of phosphate, or thevolume of the phosphate containing buffer, is low. A low flow rate canalso be preferred when the phosphate containing buffer, or the precedingsolution, is viscous or the column is fouled with adsorbed biologicalcompounds. Alternatively, the flow rate can be higher than 400 cm/hr. Insome cases, the formation of a loosely bound layer of phosphate is rapidand a high flow rate can advantageously reduce the time required forapatite regeneration.

In some embodiments, the apatite solid surface is contacted with thephosphate containing buffer in a batch format. In a batch format, thephosphate containing buffer can be applied by pouring the phosphatecontaining buffer onto the apatite solid surface, or pouring a slurry ofthe apatite solid surface into the phosphate containing buffer. Thecontact time can vary, but will generally be selected as any time thatwill reduce, eliminate, or reverse deterioration of the resin thatoccurs during apatite use (e.g., during purification, during elution, orduring cleaning/stripping).

In some embodiments, the apatite solid surface is then washed or rinsed.In other embodiments, the apatite solid surface is not washed or rinsedafter regeneration treatment with a phosphate containing buffer. One ofskill in the art can readily select a suitable wash buffer. In somecases, the resin is treated with a wash solution to remove any excesscalcium, phosphate, or hydroxide ions. Generally, the wash buffer is ata pH, composition, and concentration that does not substantially leachcomponents of the apatite surface, release accumulated hydronium ions,or generate undesirable precipitate. For example, the wash buffer can becompatible, and thus not precipitate when mixed, with the preceding andsubsequent buffer. As another example, the wash buffer can be selectedthat does not leach any loosely bound calcium layer formed during thecontacting of the apatite solid surface with the buffered calciumsolution. Suitable washing buffers can include buffer compositionstypically used for equilibration, loading, or flow through of apatite.In some cases, the apatite solid surface is washed with a low molarityphosphate buffer (e.g., phosphate at a concentration of less than about100 mM, 50 mM, 25 mM, 20 mM, 15 mM, 10 mM, or 5 mM). The pH of the washbuffer can be at least about 5, at least about 5.5, at least about 6, orat least about 6.5, 7, or 8. In some cases, a water wash is applied, andthe amounts can vary widely. A typical water wash will be at least about0.2 resin volumes, and in most cases from about 0.2 to about 1.5 or fromabout 0.2 to about 2 resin volumes.

A degree of resin regeneration can be achieved with either the bufferedcalcium solution treatment preceding the phosphate containing buffertreatment, or with the phosphate containing buffer treatment precedingthe buffered calcium solution treatment. In some embodiments, a greaterdegree of regeneration can be achieved by applying the buffered calciumsolution treatment first, followed by the phosphate containing buffertreatment. In some embodiments, a preferred degree of regeneration canbe achieved by performing one or more steps of buffered calcium solutiontreatment subsequent to, or followed by, one or more steps of phosphatecontaining buffer treatment. In some cases, one or more of multiplesteps of buffered calcium solution treatment or phosphate containingbuffer treatment are preceded by or followed by a wash.

In some embodiments, the buffered calcium solution treatment and/or thephosphate containing buffer treatment is applied after elution or flowthrough of a target analyte. For example an apatite surface can beequilibrated, contacted with a target analyte, the target analyte can beeluted or collected in the flow through, and then the regenerationprotocol can be applied. As described herein, exemplary regenerationprotocols can include, but are not limited to, those in which a bufferedcalcium solution is contacted with the apatite solid surface and then aphosphate buffer is contacted with the apatite solid surface. Exemplaryregeneration protocols can further include, but are not limited to,those in which a phosphate regeneration buffer is contacted with theapatite solid surface and then a buffered calcium regeneration solutionis contacted with the apatite solid surface. An alkaline hydroxidetreatment can be applied after the apatite is contacted with thebuffered calcium and phosphate regeneration solutions.

C. Hydroxide

The hydroxide ion treatment is applied as the last treatment step of theapatite solid surface regeneration. Any soluble form of hydroxide ioncan be used, preferably a water-soluble form is used. In some cases,alkali metal hydroxides, such as sodium or potassium hydroxide, areparticularly convenient. As in the cases of the calcium ion and thephosphate ion, the concentration and quantity of hydroxide ion solutioncan vary. Without wishing to be bound by theory, it is believed that thehydroxide interacts with the apatite solid surface, or loosely bound(e.g., non-covalently bound) calcium, phosphate, or calcium andphosphate layer(s) formed during contact with the buffered calciumsolution and/or phosphate containing buffer, to convert the looselybound (e.g., non-covalently bound) minerals into apatite, thus providinga regenerated surface. In some cases, this regenerated surface replacessome or all (or more than all) of the calcium, phosphate, or calciumphosphate lost during previous purification steps. Thus, an amount,volume, concentration, etc. of hydroxide that will reduce, eliminate, orreverse the deterioration of the resin that occurs during apatite use,can be an amount that allows for sufficient conversion of loosely boundcalcium, phosphate, or calcium phosphate to apatite. The hydroxide ioncan also clean the resin of residual proteins and contaminants and canalso serve as a sanitation or storage solution.

The hydroxide ion concentration can be from about 0.005 or 0.01 M toabout 5 M; about 0.1 M to about 4.0 M, and in many cases from about 0.3M to about 3.0 M, including 0.2 M, 0.5 M, 0.75 M, 1.0 M, 1.25 M, 1.5 M,2.0 M, or 2.5 M. Suitable volumes of hydroxide ion containing treatmentsolution range from about 1.0 to about 20.0 resin volumes, and in manycases from about 1.5 to about 10.0 resin volumes, including 2, 3, 4, 5,6, 7, 8, or 9 volumes. In some cases, a high hydroxide concentration ata volume that is less than a resin volume (e.g., less than about 0.9,0.7, 0.5 volumes) can be utilized.

Following hydroxide treatment, the resin can be washed or equilibratedwith a suitable buffer. In some cases, the resin is equilibrated, orwashed and then equilibrated, with a loading buffer. For example, theresin can be equilibrated with 10 mM phosphate buffer, pH 6.5 toequilibrate the column for protein purification. In some cases, theresin is equilibrated, or washed and then equilibrated, with a storagebuffer. For example, the resin can be equilibrated with 0.1 M NaOH, 10mM phosphate buffer and then stored.

EXAMPLES

The following examples are provided by way of illustration only and notby way of limitation. Those of skill in the art will readily recognize avariety of non-critical parameters that could be changed or modified toyield essentially the same or similar results.

Example 1—Control Experiment

This example illustrates the deterioration of a hydroxyapatite resinover a series of cycles exposing the resin to conditions that simulatethose encountered in protein separations (but without loading andeluting protein). The experiment was performed on a column measuring 20cm in length and 2.2 cm in internal diameter, with an internal volume of76 mL, and the packing was ceramic hydroxyapatite Type I in 40-micronparticles weighing approximately 48 grams, the resulting mobile phaseflow rate through the column being 250 cm/h. A series of 25 consecutivecycles were performed, each cycle consisting of the following eightsteps:

TABLE I Treatment Protocol for Simulated Cycles of Separation and ColumnRestoration Amount Column Volume Time in Step Description Mobile PhaseVolumes in mL minutes 1 Rinse Water 1 76 4.8 2 Pre-Equilibration 400 mMNaPi, pH 7.0 3 228.1 14.4 3 Equilibration 10 mM NaPi, pH 6.8 6 456.228.8 4 Equilibration 10 mM NaPi, 1.0M NaCl, pH 6.5 6 456.2 28.8 5 RinseWater 2 152.1 9.6 6 Regeneration 400 mM NaPi, pH 7.0 2 152.1 9.6 7 RinseWater 1 76 4.8 8 Sanitization 1M NaOH 2 152.1 9.6

In this protocol, Step 2 is a conditioning step to lower the pH of thecolumn following the alkali treatment of Step 8; and Steps 3 and 4expose the column to the conditions that are generally present duringcolumn equilibration, sample loading, and elution. Measurements ofparticle mass and particle strength (by uniaxial confined bulkcompression, “UCBC”) were taken before the first cycle and after thelast cycle in each of three segments of the column—the top 25%, themiddle 50%, and the bottom 25% (the mobile phase entry being at the topof the column). The results are listed in Table II below.

TABLE II Changes in Solid Phase Mass and Strength at Three ColumnLocations for Simulated Cycles of Separation and Column RestorationResin Mass Particle Strength (UCBC @ 6.00 mm) Packing After 25 PercentPercent Location Start Cycles Change N psi Change Top 25% 12.00 g 11.11g −7.42% 136.9 516 −49% Middle 50% 23.90 g 22.26 g −6.86% 156.6 590 −41%Bottom 25% 12.00 g 11.23 g −6.42% 132.05 498 −50% Total 47.90 g 44.60 g−6.89% Control: 266.4 1003

The data in Table II indicate that the resin experienced chemicalmodification as evidenced by a significant loss of mass and asignificant decline in particle strength. The overall mass loss was6.89% and was approximately uniform throughout the height of the column.The decline in particle strength was greatest at the top and bottom, butgenerally extended throughout the column as well.

Example 2

This example illustrates the result of incorporating an in situregeneration protocol without buffering the calcium regenerationsolution. The experiment was performed on a column measuring 40 cm inlength and 1.6 cm in internal diameter, with an internal volume of 80.42mL, and the packing was ceramic hydroxyapatite Type I in 40-micronparticles weighing approximately 51 grams, the resulting mobile phaseflow rate through the column being 200 cm/h. A series of 25 consecutivecycles were performed, each cycle consisting of the following elevensteps:

TABLE III Treatment Protocol for Simulated Cycles of Separation andColumn Restoration Amount Column Volume Time in Step Description MobilePhase Volumes in mL minutes 1 Pre-Equilibration 50 mM NaPi, 0.1M NaCl,pH 6.7 5.0 402.1 60 2 Equilbration 2 mM NaPi, dilute MES, 0.1M NaCl, 3.0241.3 36 pH 6.7 3 Sample 2 mM NaPi, dilute MES, dilute Tris, 7.0 563.084 0.1M NaCl, pH 6.7 4 Equipment line rinse Ultra Pure Water, line flush0.08 6.4 1.0 5 Wash 2 mM NaPi, 0.1M NaCl, pH 6.7 3.0 241.3 36 6 GradientElution Gradient 8-90%, 2 mM NaPi, 0.1M 10.0 804.2 120 NaCl, pH 6.7 → 50mM NaPi, 0.1M NaCl, pH 6.7 7 Resin clearance 50 mM NaPi, 0.1M NaCl, pH6.7 2.0 160.8 24 8 Unbuffered In-Situ 50 mM CaCl₂ 3.0 241.3 36Restoration 9 Wash 2 mM NaPi, 0.1M NaCl, pH 6.7 0.1 8.0 1.2 10Regeneration 400 mM NaPI, pH 7.0 2.0 160.8 24 11 Sanitization 1N NaOH2.0 160.8 24

In Table III, dilute MES refers to an MES concentration of at leastabout 10 mM, 15 mM, or 20 mM MES and less than about 25 mM or 30 mM MES.Dilute Tris refers to a Tris concentration of at least about 2 mM, 3 mM,4 mM, 5 mM, 10 mM, or 15 mM Tris and less than about 20 mM, or 25 mMTris.

As in Example 1, 25 cycles were performed with measurements of particlemass and particle strength taken before the first cycle and after thelast cycle. The measurements indicate that particle mass for the entirecolumn increased by 7.18% and particle strength decreased by 20%, overthe course of the 25 cycles. Thus, although some regeneration wasachieved, as evidenced by the increase in mass and the smaller loss ofparticle strength compared to Example 1, the apatite is stillsignificantly degraded.

Example 3

This example illustrates the result of incorporating an in situregeneration protocol that includes a buffered calcium regenerationsolution. The experiment was performed on a column measuring 20 cm inlength and 2.2 cm in internal diameter, with an internal volume of 76.03mL, and the packing was ceramic hydroxyapatite Type I in 40-micronparticles weighing approximately 48 grams, the resulting mobile phaseflow rate through the column being 250 cm/h. A series of 25 consecutivecycles were performed, each cycle consisting of the following ninesteps:

TABLE IV Treatment Protocol Amount Column Volume Time in StepDescription Mobile Phase Volumes in mL minutes 1 Rinse Water 1.0 76.04.8 2 Pre-Equilibration 400 mM NaPi, pH 7.0 3.0 228.1 14.4 3Equilibration 5 mM NaPi, 5 mM MES, pH 6.5 6.0 456.2 28.8 4 Elution 10 mMNaPi, 25 mM MES, 1.0M 6.0 456.2 28.8 NaCl, pH 6.5 5 Buffered In-Situ 50mM CaCl2, 10 mM BES, pH 7.5 3.0 228.1 14.4 Restoration 6 Rinse Water 2.0152.1 9.6 7 Regeneration 400 mM NaPi, pH 7.0 2.0 152.1 9.6 8 Rinse Water1.0 76.0 4.8 9 Sanitization 1N NaOH 2.0 152.1 9.6

Hydroxyapatite obtained from a column operated for 25 cycles using theprotocol in Table IV gained 4.74% in mass and increased 30% in particlestrength compared to an unused hydroxyapatite control. These resultsdemonstrate that the use of a buffered calcium solution, followed byapplication of a phosphate buffer and then a hydroxide provides asignificant and surprising degree of regeneration, as evidenced by bothan increase in mass and a significant increase in particle strength.

Example 4

This example illustrates the result of incorporating an in situregeneration protocol that includes a buffered calcium regenerationsolution into an apatite based purification protocol that utilizesalkali metal salt step gradient elution. The experiment was performed ona column measuring 20 cm in length and 2.2 cm in internal diameter, withan internal volume of 76.03 mL, and the packing was ceramichydroxyapatite Type I in 40-micron particles weighing approximately 48grams, the resulting mobile phase flow rate through the column being 400cm/h. A series of 30 consecutive cycles were performed, each cycleconsisting of the following eight steps:

TABLE V Treatment Protocol Amount Column Volume Time in Step DescriptionMobile Phase Volumes in mL minutes 1 Pre-Equilibration 400 mM NaPi, pH7.0 3.0 228.1 9 2 Equilibrium-Load-Wash 10 mM NaPi, pH 6.5 10.0 760.3 303 Elution 10 mM NaPi, 1.0M NaCl, pH 6.5 6.0 456.2 18 4 Rinse 10 mM NaPi,pH 6.5 0.2 15.2 0.6 5 Buffered In-Situ 50 mM CaCl₂•2H₂O, 100 mM MES, 3.0228.1 9 Restoration pH 7 6 Rinse 10 mM NaPi, pH 6.5 0.2 15.2 0.6 7Regeneration 400 mM NaPi, pH 7.0 2.0 152.1 6 8 Sanitization 1N NaOH 2.0152.1 6

Hydroxyapatite obtained from a column operated for 30 cycles using theprotocol in Table V gained 20.8% in mass and increased 13% in particlestrength compared to an unused hydroxyapatite control. These resultsdemonstrate that the use of a buffered calcium solution provides asignificant and surprising degree of regeneration even when a highconcentration of alkali metal salt is utilized in the elution step.

Example 5

This example illustrates the result of incorporating an in situregeneration protocol that includes a buffered calcium regenerationsolution into an apatite based purification protocol that utilizesalkali metal salt step gradient elution. The experiment was performed ona column measuring 20 cm in length and 1.6 cm in internal diameter, withan internal volume of 40.21 mL, and the packing was ceramichydroxyapatite Type I in 40-micron particles weighing approximately 25grams, the resulting mobile phase flow rate through the column being 400cm/h. A series of 25 consecutive cycles were performed, each cycleconsisting of the following eight steps:

TABLE VI Treatment Protocol Amount Column Volume Time in StepDescription Mobile Phase Volumes in mL minutes 1 Pre-Equilibration 400mM NaPi, pH 7.0 3.0 120.6 9 2 Equilibrium-Load-Wash 10 mM NaPi, pH 6.510.0 402.1 30 3 Elution 10 mM NaPi, 1.0M NaCl, pH 6.5 6.0 241.3 18 4Rinse 10 mM NaPi, pH 6.5 0.2 8.0 0.6 5 Buffered In-Situ 50 mMCaCl₂•2H₂O, 100 mM MES, 1.1 44.2 3.3 Restoration pH 7 6 Rinse 10 mMNaPi, pH 6.5 0.2 8.0 0.6 7 Regeneration 400 mM NaPi, pH 7.0 2.0 80.4 6 8Sanitization 1N NaOH 2.0 80.4 6

Hydroxyapatite obtained from a column operated for 25 cycles using theprotocol in Table VI gained 12.6% in mass and increased 12% in particlestrength compared to an unused hydroxyapatite control. The resultsdemonstrate that significant regeneration is achieved even when a highconcentration of alkali metal salt is utilized in the elution step.Moreover, the substantial regeneration is achieved with a low volume(1.1 column volumes) of buffered calcium solution.

Example 6

This example illustrates the result of incorporating an in situregeneration protocol that includes a buffered calcium regenerationsolution into an alkali metal salt supplemented phosphate gradient basedapatite based purification protocol. The experiment was performed on acolumn measuring 40 cm in length and 2.2 cm in internal diameter, withan internal volume of 152.05 mL, and the packing was ceramichydroxyapatite Type I in 40-micron particles weighing approximately 96grams, the resulting mobile phase flow rate through the column being 350cm/h. A series of 30 consecutive cycles were performed, each cycleconsisting of the following eleven steps:

TABLE VII Treatment Protocol Amount Column Volume Time in StepDescription Mobile Phase Volumes mL/min in mL minutes 1 Equilibration 150 mM NaPi, 0.1M NaCl, pH 6.7 1 11.09 152.1 13.7 2 Equilibration 1 50 mMNaPi, 0.1M NaCl, pH 6.7 4 22.17 608.2 27.4 3 Equilibration 2 2 mM NaPi,dilute MES, 0.1M NaCl, 3 22.17 456.2 20.6 pH 6.7 4 Sample 2 mM NaPi,dilute MES, 0.1M NaCl, 7 22.17 1064.4 48 pH 6.7 5 Wash 2 mM NaPi, 0.1MNaCl, pH 6.5 3 22.17 456.2 20.6 6 Gradient 10% Equil-1, 90% 0.1M NaCl →10 22.17 1520.5 68.6 90% Equil-1, 10% 0.1M NaCl 7 Equilibration 1 50 mMNaPi, 0.1M NaCl, pH 6.7 2 22.17 304.1 13.7 8 Equilibration 2 2 mM NaPi,dilute MES, 0.1M NaCl, 0.2 22.17 30.41 1.4 pH 6.7 9 In-Situ Restoration50 mM CaCl₂, 100 mM MES, pH 7.0 3 11.09 456.2 41.1 10 Equilibration 2 2mM NaPi, dilute MES, 0.1M NaCl, 0.2 11.09 30.41 2.7 pH 6.7 11Regeneration 400 mM NaPi, pH 7.0 2 11.09 304.1 27.4 12 Sanitization 1MNaOH 2 11.09 304.1 27.4

In Table VII, dilute MES refers to an MES concentration of at leastabout 10 mM, 15 mM, or 20 mM MES and less than about 25 mM or 30 mM MES.Dilute Tris refers to a Tris concentration of at least about 2 mM, 3 mM,4 mM, 5 mM, 10 mM, or 15 mM Tris and less than about 20 mM, or 25 mMTris.

Hydroxyapatite obtained from a column operated for 30 cycles using theprotocol in Table VII gained 18% in mass and increased 45% in particlestrength compared to an unused hydroxyapatite control. Again, theprotocol provides a significant and surprising degree of apatiteregeneration as evidenced by a gain in both apatite mass and apatiteparticle strength.

Example 7

This example illustrates the result of incorporating an in situregeneration protocol that includes a buffered calcium regenerationsolution into a phosphate gradient based apatite based purificationprotocol. The experiment was performed on a column packed with 40-micronceramic hydroxyapatite Type I particles, the resulting mobile phase flowrate through the column being 250 cm/h. A series of consecutive cycleswere performed, each cycle consisting of the steps outlined in TableVIII.

TABLE VIII Treatment Protocol Column Step Protocol Reagent Volumes 1Equilibration 1 5.0 2 Equilibration 2 3.0 3 Sample 7.0 4 Wash 3.0 5Gradient 10-90% 0.1M NaCl > 0.1M 10.0  NaCl/phosphate solution 6 Wash2.0 7 Line rinse 0.4 8 Calcium In-Situ Restoration solution Varies 9Line rinse 0.4 10 400 mM NaPi, pH 7.0 2.0 11 1M NaOH 2.0

The “Calcium ISR solution” listed in Table VIII was varied for eachexperiment as described in Table IX.

TABLE IX Calcium ISR (in situ regeneration) Legend dashed dashed dasheddashed black purple orange orange blue blue light blue light blue redExperiment 5897-045 5897-072/Col17 Baseline 6141-034 6141-035 6141-0376141-038 6141-040 6141-041 6045-023 Cycles 20 30 24 24 24 24 24 24 24[CaCl2] mM 50.0 10.0 25.0 25.0 25.0 5.74 10.2 50.0 [MES] mM 100 100 100100 20 100 pH 7.00 7.00 5.60 6.20 5.40 5.30 7.00 CV 3.00 0 1.10 1.101.10 1.10 3.00 3.00 1.10 [PO4] mM 23 23

Table X provides further experimental details for a test of the relativeselectivity of standard proteins on four lots of CHT Type 1, 40 μmcontrol media.

TABLE X Relative Selectivity Protocol CV 2.03 FR, cm/hr 250 Column ID cmLength ml/min 0.692 5.4 1.57 Area cm² 0.3761 Protiens in DH2O mg 10 mlOvalbumin 100 Myoglogin 75 α-chymotrysinogen a 70 Cytochrome C 70 StepReagent CV Sanitize 1N NaOH 3.0 Regen 400 mM NaPi, pH 6.8 4.0 Equil 5 mMNaPi, pH 6.8 15.0 Inject Protein Load 0.1 Equil 5 mM NaPi, pH 6.8 1.0Gradient Gradient 0 −> 75 15.0 Regen Regen 400 mM NaPi, pH 6.8 3.0

Results from the performing the purification protocol of Table X areillustrated in FIG. 1. The apatite media was then cycled with a bufferedregeneration solution containing 50 mM calcium and 100 mM MES, or anunbuffered calcium regeneration solution containing 50 mM calcium. Therelative selectivity of alpha-chymotrypsinogen A changes on media cycledwith 50 mM calcium buffered with 100 mM MES solution at pH 7.0 (red), asshown in FIG. 2. As also shown in FIG. 2, the selectivity is maintainedafter cycling with an unbuffered in situ regeneration (ISR) solution(black dotted) although a loss in resolution is noted relative to thecontrol (green).

FIG. 3 shows that the relative selectivity of alpha-chymotrypsinogen Ais maintained when cycled with an the ISR solution of 25 mM calciumchloride buffered at pH 6.2 with 100 mM MES (dashed blue) or buffered atpH 5.6 (blue). The resolution between myoglobin andalpha-chymotrypsinogen A at pH 6.2 or 5.6 is affected but not theirrelative selectivity. Their relative selectivity is similar to thebaseline protocol resin (5897-045 BL) absent the buffered ISR solution(purple), control resin (green) and unbuffered ISR resin (5897-072/Col17(dashed black). The relative selectivity of resin cycled with an ISRsolution that is 50 mM calcium buffered at pH 7.0 with 100 mM MES (red)is lost.

FIG. 4 shows that the relative selectivity of alpha-chymotrypsinogen Ais maintained similarly to the control (green) when the concentration ofcalcium in the buffered ISR solution during cycling is reduced to 10 mM(orange) or 25 mM (light blue) while maintaining the buffer at pH 7.0with 100 mM MES. This is not the case for the 50 mM calcium solutionunder the same buffering conditions (red). The resolution betweenmyoglobin and alpha-chymotrypsinogen A is reduced to that observed withunbuffered ISR (dotted black) but the selectivity for each ismaintained.

FIG. 5 shows that the relative selectivity is unaffected by cycling witha regeneration solution containing a low concentration of calcium (5.74mM) buffered at pH 5.40 with 20 mM MES or 23 mM phosphate (light blue).In addition 10.2 mM calcium containing 23 mM phosphate maintainsselectivity but has decreased resolution (dashed light blue). Both ofthese conditions are comparable to 25 mM calcium buffered at pH 5.6 with100 mM MES (blue) and unbuffered ISR (dashed black).

The mass of the apatite media increased for all cycling experimentsconducted with in situ regeneration (ISR), as depicted in Table XI. Thebaseline media (5897-045 BL) decreased in mass. With exception ofexperiment 5897-017/Col17 the loss in media strength did not declinebelow the value in N/mm for the other experiments.

TABLE XI Results Experiment 5897-045 5897-072/Col17 Baseline 6141-0346141-035 6141-037 6141-038 6141-040 6141-041 Cycles 20 30 24 24 24 24 2424 Mass change 3.92 −2.64 0.14 0.96 0.50 1.03 0.35 0.57 % Change 4.09−2.76 1.10 7.58 3.95 8.13 2.76 4.50 Media Strength Average 4.90 4.033.41 2.78 2.90 2.77 3.48 2.74 STDEV 0.582 0.235 0.186 0.230 0.103 0.1010.240 0.250 RSD 0.119 0.058 0.054 0.083 0.035 0.037 0.069 0.091 N/mm20.4 24.8 29.4 36.0 34.4 36.1 28.8 36.5

The results indicate that both the calcium concentration and the pH ofbuffered calcium solution affect the relative selectivity of standardproteins. Acceptable performance can be achieved with a buffered calciumsolution at, e.g., a pH of at least about 5.3, a calcium concentrationof at least about 5 mM and a phosphate or zwitterionic bufferconcentration of between about 20 mM and 100 mM.

All patents, patent applications, and other published referencematerials cited in this specification are hereby incorporated herein byreference in their entirety.

What is claimed is:
 1. A method of purifying a target analyte with anapatite solid surface, the method comprising: (a) contacting the apatitesolid surface with the target analyte, thereby separating the targetanalyte from one or more contaminants; (b) collecting the targetanalyte, wherein the contacting, the collecting, or both causedeterioration of resin mass or particle strength; and (c) after thecollecting and before subsequent loading of additional target analyte,regenerating the apatite solid surface, the regenerating comprising: (i)contacting the apatite solid surface with a buffered calcium solutioncomprising a calcium ion at a concentration of at least 5 mM and azwitterionic buffer, wherein the ratio of zwitterionic bufferconcentration to calcium ion concentration is at least 2, and the pH ofthe buffered calcium solution is at least 5; (ii) after (i), contactingthe apatite solid surface with a phosphate buffered solution at a pH ofat least 6.5; and (iii) after (ii), contacting the apatite solid surfacewith a solution comprising an hydroxide, wherein step (c) reduces,eliminates, or reverses the deterioration of the resin mass or particlestrength of said apatite solid surface.
 2. The method of claim 1,wherein (a) comprises binding the target analyte to the apatite solidsurface, and (b) comprises eluting the target analyte from the apatitesolid surface.
 3. The method of claim 1, wherein (a) comprisescontacting the apatite solid surface with the target analyte, therebyflowing the target analyte through the apatite solid surface, and (b)comprises collecting the target analyte in the flow through.
 4. Themethod of claim 1, wherein the zwitterionic buffer is a sulfonic acidcontaining buffer.
 5. The method of claim 4, wherein the sulfonic acidcontaining buffer is MES, PIPES, ACES, MOPSO, MOPS, BES, TES, HEPES,DIPSO, TAPS, TAPSO, POPSO, HEPPSO, EPPS, CAPS, CAPSO, or CHES.
 6. Themethod of claim 5, wherein the sulfonic acid containing buffer is MES.7. The method of claim 1, wherein the calcium ion concentration is lessthan 50 mM.
 8. The method of claim 1, wherein the calcium ionconcentration is at least 25 mM.
 9. The method of claim 1, wherein theratio of zwitterionic buffer concentration to calcium ion concentrationis at least 2.5.
 10. The method of claim 1, wherein the buffered calciumsolution comprises calcium chloride or calcium nitrate.
 11. The methodof claim 1, wherein the buffered calcium solution is at a pH of at least5.3.
 12. The method of claim 1, wherein the buffered calcium solution isat a pH of between 5.3 and
 7. 13. The method of claim 1, wherein thephosphate buffered solution comprises a solution containing from 0.1 Mto 1.0 M phosphate at a pH of from 6.5 to
 8. 14. The method of claim 13,wherein the phosphate buffered solution comprises 400 mM phosphate at apH of 7.0.
 15. The method of claim 1, wherein the hydroxide comprises analkaline hydroxide.
 16. The method of claim 15, wherein the alkalinehydroxide comprises sodium or potassium hydroxide.
 17. The method ofclaim 1, wherein the regenerating reverses or eliminates degradation ofthe column that occurs during protein purification or column cleaningsteps.
 18. The method of claim 1, wherein the contacting of the apatitesolid surface with the phosphate buffered solution comprising phosphateat a pH of at least 6.5 further comprises: contacting the apatite solidsurface with a solution comprising phosphate at a concentration of 10 mMor less at a pH of at least 6.5; and then contacting the apatite solidsurface with a solution comprising phosphate at a concentration of atleast 100 mM at a pH of at least 6.5.
 19. The method of claim 1, whereinthe regenerating consists of step (c)(i), followed by contacting theapatite with a wash, then steps (c)(ii) and, then (c)(iii).